Sulfotransferase 1e1 sequence variants

ABSTRACT

Isolated sulfotransferase nucleic acid molecules that include a nucleotide sequence variant and nucleotides flanking the sequence variant are described, as well as sulfotransferase allozymes. Methods for determining if a mammal is predisposed to cancer also are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/858,820, filedAug. 18, 2010, which is a continuation of U.S. Ser. No. 11/861,075,filed Sep. 25, 2007, which is a divisional of U.S. Ser. No. 10/354,763,filed Jan. 30, 2003, now U.S. Pat. No. 7,288,641, which claims priorityfrom U.S. Provisional Application Ser. No. 60/353,066, filed Jan. 30,2002.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersGM35720 and GM61388, awarded by the National Institutes of Health, andgrant number DAMD17-99-1-9281, awarded by the Department of Defense. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The invention relates to sulfotransferase nucleic acid and amino acidsequence variants.

BACKGROUND

Sulfate conjugation, i.e., sulfonation, is an important pathway in thebiotransformation of many neurotransmitters, hormones, drugs and otherxenobiotics, and is catalyzed by cytosolic sulfotransferase enzymesdesignated “SULT.” SULT enzymes are encoded by a gene superfamily, whichin mammals is divided into two families: SULT1, or phenol SULTs, andSULT2, or hydroxysteroid SULTs. The SULT1 and SULT2 families share atleast 45% amino acid sequence identity, while members of subfamilieswithin each family share at least 60% amino acid sequence identity.SULT1 subfamilies include the phenol (1A), thyroid hormone (1B),hydroxyarylamine (1C), and estrogen (1E) subfamilies. SULT2 subfamiliesinclude two hydroxysteroid SULTs, 2A1 and 2B1.

Members of the SULT1E subfamily catalyze the sulfate conjugation ofestrogens. Human SULT1E1, for example, catalyzes the transfer of asulfonate group from the sulfonate donor 3′-phosphoadenosine5′-phosphosulfate (PAPS) to the hydroxyl group of an estrogen molecule.SULT1E1 is expressed in adult human liver, small intestine, adrenalcortex, adrenal medulla, mammary gland, ovary, endometrium, prostate,testis, and epididymus. It is also expressed in fetal lung, liver, andkidney.

SUMMARY

The invention is based on the discovery of sequence variants that occurin both coding and non-coding regions of SULT1E1 nucleic acids. CertainSULT1E1 nucleotide sequence variants encode SULT1E1 enzymes that areassociated with individual differences in enzymatic activity. OtherSULT1E1 sequence variants in non-coding regions of the SULT1E1 nucleicacid may alter regulation of transcription and/or splicing of theSULT1E1 nucleic acid. Discovery of these sequence variants allowsindividual differences in the sulfate conjugation of drugs and otherxenobiotics in humans to be assessed such that particular treatmentregimens can be tailored to an individual based on the presence orabsence of one or more sequence variants. Identification of SULT1E1sequence variants also allows predisposition to hormone dependentdiseases or chemical carcinogenesis to be assessed in individuals.

In one aspect, the invention features an isolated nucleic acid moleculecontaining a SULT1E1 nucleic acid sequence, where the nucleic acidmolecule is at least ten nucleotides in length, and where the SULT1E1nucleic acid sequence contains a nucleotide sequence variant relative toSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQID NO:6. The nucleotide sequence variant can be at a position selectedfrom the group consisting of position −232, −190, 64, 95, 237, 459, or758 relative to the adenine of the SULT1E1 translation initiation codon.The nucleotide sequence variant relative to the adenine of the SULT1E1translation initiation codon can be selected from the group consistingof an adenine substitution for guanine at position −232, a guaninesubstitution for cytosine at position −190, a thymine substitution forguanine at position 64, a thymine substitution for cytosine at position95, a cytosine substitution for thymine at position 237, a thyminesubstitution for cytosine at position 459, and an adenine substitutionfor cytosine at position 758.

The nucleotide sequence variant can be at a position selected from thegroup consisting of position −20 relative to the guanine in the spliceacceptor site of intron 1 (e.g., a thymine substitution for adenine);position 22 relative to the guanine in the splice donor site of intron 2(e.g., a cytosine substitution for thymine); position −80 relative tothe guanine in the splice acceptor site of intron 3 (e.g., a guaninesubstitution for adenine); and position 69 or 139 relative to theguanine in the splice donor site of intron 4 (e.g., a thyminesubstitution for adenine at position 69 or a thymine substitution foradenine at position 139). The nucleotide sequence variant can be at aposition selected from the group consisting of position −23 relative tothe guanine in the splice acceptor site of intron 4 (e.g., a guaninesubstitution for adenine); position 55 relative to the guanine in thesplice donor site of intron 5 (e.g., a thymine substitution forcytosine); position 55 relative to the guanine in the splice donor siteof intron 6 (e.g., a guanine deletion); position −39 relative to theguanine in the splice acceptor site of intron 6 (e.g., a cytosinesubstitution for thymine); and position −63 relative to the guanine inthe splice acceptor site of intron 7 (e.g., a guanine substitution forthymine). The nucleotide sequence variant can be a substitution or adeletion.

In another aspect, the invention features an isolated nucleic acidencoding a SULT1E1 polypeptide, where the polypeptide contains a SULT1E1amino acid sequence variant relative to the amino acid sequence of SEQID NO:7. The amino acid sequence variant can be at a residue selectedfrom the group consisting of 22, 32, and 253.

The invention also features an isolated SULT1E1 polypeptide, wherein thepolypeptide contains a SULT1E1 amino acid sequence variant relative tothe amino acid sequence of SEQ ID NO:7. The amino acid sequence variantcan be at a residue selected from the group consisting of 22, 32, and253. The amino acid sequence variant at residue 22 can be tyrosine, theamino acid sequence variant at residue 32 can be valine, and the aminoacid sequence variant at residue 253 can be histidine. The activity ofthe polypeptide can be altered relative to a wild type SULT1E1polypeptide.

In another aspect, the invention features an article of manufacturecontaining a substrate, where the substrate contains a population of theisolated SULT1E1 nucleic acid molecules described above. The substratecan contain a plurality of discrete regions, where each region containsa different population of isolated SULT1E1 nucleic acid molecules, andwhere each population of molecules contains a different SULT1E1nucleotide sequence variant.

In yet another aspect, the invention features a method for determiningif a mammal is predisposed to cancer. The method can include: a)obtaining a biological sample from the mammal; and b) detecting thepresence or absence of a SULT1E1 nucleotide sequence variant in thesample, where predisposition to cancer is determined based on thepresence or absence of the variant. The method can further includedetecting the presence or absence of a plurality of the SULT1E1nucleotide sequence variants in the sample to obtain a variant profileof the mammal, where predisposition to cancer is determined based on thevariant profile. The cancer can be an estrogen responsive cancer (e.g.,breast cancer or endometrial cancer).

In another aspect, the invention features a method for assisting amedical or research professional. The method can include: a) obtaining abiological sample from a mammal; and b) detecting the presence orabsence of a plurality of SULT1E1 nucleotide sequence variants in thesample to obtain a variant profile of the mammal. The method can furtherinclude communicating the profile to a medical or research professional.

In another aspect, the invention features an isolated nucleic acidmolecule containing a SULT1E1 nucleic acid sequence, where the nucleicacid molecule is at least ten nucleotides in length, and where theSULT1E1 nucleic acid sequence contains at least two nucleotide sequencevariants relative to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, or SEQ ID NO:6. The nucleotide sequence variant can be at aposition selected from the group consisting of: a) position −232, −190,−64, 64, 95, 237, 459, or 758 relative to the adenine of the SULT1E1translation initiation codon; b) position 69 relative to the splicedonor site of intron 1; c) position −73 or −20 relative to the guaninein the splice acceptor site of intron 1; d) position 22 relative to theguanine in the splice donor site of intron 2; e) position −137 or −80relative to the guanine in the splice acceptor site of intron 3; f)position 69 or 139 relative to the guanine in the splice donor site ofintron 4; g) position −23 relative to the guanine in the splice acceptorsite of intron 4; h) position 55 relative to the guanine in the splicedonor site of intron 5; i) position −10 relative to the guanine in thesplice acceptor site of intron 5; j) position 55 relative to the guaninein the splice donor site of intron 6; k) position −39 relative to theguanine in the splice acceptor site of intron 6; and 1) position −121 or−63 relative to the guanine in the splice acceptor site of intron 7.

In still another aspect, the invention features an isolated nucleic acidmolecule containing a SULT1E1 nucleic acid sequence, where the nucleicacid molecule is at least ten nucleotides in length, and wherein theSULT1E1 nucleic acid sequence has at least 99% sequence identity to aregion of SEQ ID NO:6. Nucleotide 64 relative to the adenine of theSULT1E1 translation initiation codon can be a thymine, nucleotide 95relative to the adenine of the SULT1E1 translation initiation codon canbe a thymine, or nucleotide 758 relative to the adenine of the SULT1E1translation initiation codon can be an adenine. The region can beselected from the group consisting of: a) nucleotides 1 to 100 of SEQ IDNO:6 relative to the adenine of the SULT1E1 translation initiationcodon; b) nucleotides 50 to 150 of SEQ ID NO:6 relative to the adenineof the SULT1E1 translation initiation codon; and c) nucleotides 700 to800 of SEQ ID NO:6 relative to the adenine of the SULT1E1 translationinitiation codon.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is the genomic sequence of the reference human SULT1E1 (SEQ IDNOS:1-5). Intron, 5′-flanking region and 3′-flanking region sequencesare in lower case letters. Primers used for resequencing are underlined.Single nucleotide polymorphisms (SNPs) are bold, in italics, and areunderlined. The position and nature of each SNP, insertion, and deletionis indicated proximal to the markings described. Exon sequences arebolded and in upper case letters. The start codon also is italicized.

FIGS. 2A and 2B are the cDNA and amino acid sequences of the referenceSULT1E1, respectively (SEQ ID NOS:6 and 7). Start and stop codons arebold and underlined.

FIG. 3 is a schematic showing locations of polymorphisms within theSULT1E1 sequence.

FIG. 4 is a graph plotting the activity levels of three SULT1E1allozymes as a percent of the wild-type SULT1E1 enzyme activity.

FIG. 5 is a graph plotting quantitative results of a western blot assayin which the amount of immunoreactive protein for each of three SULT1E1allozymes was compared with the amount of immunoreactive protein for thewild-type SULT1E1 enzyme.

DETAILED DESCRIPTION

The invention features SULT1E1 nucleotide and amino acid sequencevariants. SULT1E1 catalyzes the transfer of a sulfonate group to steroidhormone molecules such as estrone, estradiol, catecholestrogens, andmethoxyestradiol. For example, SULT1E1 can catalyze the transfer of asulfonate group from PAPS to estradiol to form estradiol 3-O-sulfate.Sulfonation inactivates estrogen molecules, as sulfonated estrogens haveno effect on the estrogen receptor. Therefore, increased sulfonation ofestrogens may be a protective mechanism against estrogen responsivecancers such as breast cancer and endometrial cancer. SULT1E1 has beenshown to modulate local estrogen levels in a breast cancer cell line.Without being bound by a particular mechanism, a loss or down-regulationof SULT1E1 could increase the growth stimulating effect of estrogen andcontribute to the process of tumor initiation and promotion in breastepithelium. Thus, detecting sulfotransferase nucleic acid and amino acidsequence variants can be useful for diagnosing cancer as well as fordetermining a predisposition for cancer.

Furthermore, sulfation can detoxify compounds, as the resulting ionized,organic sulfates are more readily excreted than unsulfated compounds.Thus, detecting sulfotransferase nucleic acid and amino acid sequencevariants can facilitate the prediction of therapeutic efficacy andtoxicity of drugs on an individual basis, as well as the ability tobiotransform certain hormones and neurotransmitters.

Nucleic Acid Molecules

The invention features isolated nucleic acids that include a SULT1E1nucleic acid sequence. The SULT1E1 nucleic acid sequence includes anucleotide sequence variant and nucleotides flanking the sequencevariant. As used herein, “isolated nucleic acid” refers to a nucleicacid that is separated from other nucleic acid molecules that arepresent in a mammalian genome, including nucleic acids that normallyflank one or both sides of the nucleic acid in a mammalian genome (e.g.,nucleic acids that encode non-SULT1E1 proteins). The term “isolated” asused herein with respect to nucleic acids also includes anynon-naturally-occurring nucleic acid sequence sincenon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone or both of the nucleic acid sequences normally found immediatelyflanking that DNA molecule in a naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, aDNA molecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as recombinant DNA that is incorporated into a vector,an autonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a recombinant DNA moleculethat is part of a hybrid or fusion nucleic acid. A nucleic acid existingamong hundreds to millions of other nucleic acids within, for example,cDNA libraries or genomic libraries, or gel slices containing a genomicDNA restriction digest, is not to be considered an isolated nucleicacid.

Nucleic acids of the invention are at least about 8 nucleotides inlength. For example, a nucleic acid can be about 8, 9, 10-20 (e.g., 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length), 20-50,50-100, or greater than 100 nucleotides in length (e.g., greater than150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 nucleotides inlength). Nucleic acids of the invention can be in a sense or anantisense orientation, can be complementary to the SULT1E1 referencesequence, and can be DNA, RNA, or nucleic acid analogs. Nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′-hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six membered, morpholino ring, orpeptide nucleic acids, in which the deoxyphosphate backbone is replacedby a pseudopeptide backbone and the four bases are retained. See,Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195;and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, thedeoxyphosphate backbone can be replaced with, for example, aphosphorothioate or phosphorodithioate backbone, a phosphoroamidite, oran alkyl phosphotriester backbone.

As used herein, “nucleotide sequence variant” refers to any alterationin the SULT1E1 reference sequence, and includes variations that occur incoding and non-coding regions, including exons, introns, anduntranslated sequences. Nucleotides are referred to herein by thestandard one-letter designation (A, C, G, or T). Variations can includesingle nucleotide substitutions, deletions of one or more nucleotides,and insertions of one or more nucleotides. Reference SULT1E1 nucleicacid sequences are provided in FIG. 1 (SEQ ID NOS:1-5) and in GenBank(Accession Nos: U20515-U20521). The reference SULT1E1 messenger RNA(mRNA) including the reference SULT1E1 cDNA is provided in FIG. 2A (SEQID NO:6) and in GenBank (Accession No: U08098), and the correspondingSULT1E1 amino acid sequence is provided in FIG. 2B (SEQ ID NO:7) and inGenBank (Accession No. U08098). The nucleic acid and amino acidreference sequences also are referred to herein as “wild type.”

As used herein, “untranslated sequence” includes 5′- and 3′-flankingregions that are outside of the mRNA as well as 5′- and 3′-untranslatedregions (5′-UTR or 3′-UTR) that are part of the mRNA, but are nottranslated. Positions of nucleotide sequence variants in 5′-untranslatedsequences are designated as “−X” relative to the “A” in the translationinitiation codon; positions of nucleotide sequence variants in thecoding sequence and 3′-untranslated sequence are designated as “+X” or“X” relative to the “A” in the translation initiation codon. Nucleotidesequence variants that occur in introns are designated as “+X” or “X”relative to “G” in the splice donor site (GT) or as “−X” relative to the“G” in the splice acceptor site (AG).

In some embodiments, a SULT1E1 nucleotide sequence variant encodes aSULT1E1 polypeptide having an altered amino acid sequence. The term“polypeptide” refers to a chain of at least four amino acid residues(e.g., 4-8, 9-12, 13-15, 16-18, 19-21, 22-100, 100-150, 150-200, 200-300residues, or a full-length SULT1E1 polypeptide). SULT1E1 polypeptidesmay or may not have sulfotransferase catalytic activity, or may havealtered activity relative to the reference SULT1E1 polypeptide.Polypeptides that do not have activity or have altered activity can beuseful for diagnostic purposes (e.g., for producing antibodies havingspecific binding affinity for variant sulfotransferase polypeptides).

Corresponding SULT1E1 polypeptides, irrespective of length, that differin amino acid sequence from the reference SULT1E1 polypeptide arereferred to herein as allozymes. For example, a SULT1E1 nucleic acidsequence that includes a thymine at nucleotide 64 relative to theadenine in the SULT1E1 translation initiation site encodes a SULTpolypeptide having a tyrosine at amino acid residue 22. This polypeptide(Asp22Tyr) would be considered an allozyme with respect to the referenceSULT1E1 polypeptide that contains an aspartic acid at amino acid residue22. Additional non-limiting examples of SULT1E1 sequence variants thatalter amino acid sequence include variants at nucleotides 95 and 758relative to the adenine in the SULT1E1 translation initiation site. Forexample, a SULT1E1 nucleic acid molecule can include a thymine atnucleotide 95 and encode a SULT1E1 polypeptide having a valine residueat amino acid 32 in place of an alanine residue (Ala32Val), or can havean adenine at nucleotide 758 and encode a SULT1E1 polypeptide having ahistidine at amino acid 253 in place of a proline (Pro253His). Inaddition, a SULT1E1 nucleic acid can encode an allozyme having two ormore amino acid variants, e.g., the nucleic acid can encode apolypeptide having two or more amino acid changes at residues 22, 32,and 253.

SULT1E1 allozymes as described above are encoded by a series ofsulfotransferase alleles. These alleles represent nucleic acid sequencescontaining sequence variants, typically multiple sequence variants,within coding and non-coding sequences. Representative examples ofsingle nucleotide variants are described above. Table 2 includes threepolymorphisms that result in SULT1E1 alleles encoding the SULT allozymesAsp22Tyr, Ala32Val, and Pro253His. The number of alleles and allozymesfor SULT1E1 indicates the potential complexity of SULT pharmacogenetics.Such complexity emphasizes the need for determining single nucleotidevariants, (i.e., single nucleotide polymorphisms, SNPs) as well ascomplete haplotypes (i.e., the set of alleles on one chromosome or apart of a chromosome) of patients. See Table 4 for haplotypes ofSULT1E1.

Certain SULT1E1 nucleotide sequence variants do not alter the amino acidsequence. Such variants, however, could alter regulation oftranscription as well as mRNA stability. SULT1E1 variants can occur inintron sequences, for example, within intron 1, 2, 3, 4, 5, 6, or 7. Inparticular, the nucleotide sequence variant can include a guaninesubstitution at nucleotide 69, a cytosine substitution at nucleotide−73, or a thymine substitution at nucleotide −20 of intron 1. Intron 2variants can include a cytosine substitution at nucleotide 22. Intron 3variants can include a guanine substitution at nucleotide −137 ornucleotide −80. Intron 4 variants can include a thymine substitution atnucleotide 69 or nucleotide 139, or a guanine substitution at nucleotide−23. Intron 5 sequence variants can include a thymine substitution atnucleotide 55 or a guanine substitution at nucleotide −10. Intron 6sequence variants can include a guanine deletion at nucleotide 55 or acytosine substitution at nucleotide −39. Intron 7 variants can include athymine substitution at nucleotide −121 or a guanine substitution atnucleotide −63.

SULT1E1 nucleotide sequence variants that do not change the amino acidsequence also can be within an exon or in 5′- or 3′-untranslatedsequences. For example, a nucleotide sequence variant can include anadenine substitution at nucleotide −64 of exon 1, a cytosinesubstitution at nucleotide 237 of exon 3, or a thymine substitution atnucleotide 459 of exon 5. In addition, the 5′-flanking region of SULT1E1can include an adenine substitution at nucleotide −232 or a guaninesubstitution at nucleotide −190 relative to the adenine in the SULT1E1transcription initiation site. Nucleotides −232 and −190 are atpositions 643 and 685, respectively, relative to the first nucleotide ofSEQ ID NO:1 (see FIG. 1).

Isolated nucleic acid molecules provided herein can contain a SULT1E1nucleic acid sequence that is at least ten nucleotides in length andincludes a nucleotide sequence variant relative to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In someembodiments, a SULT1E1 nucleic acid can include at least two (e.g., two,three, four, five, or more than five) nucleotide sequence variants.Examples of SULT1E1 nucleotide sequence variants are provided herein.

In some embodiments, nucleic acid molecules of the invention can have atleast 97% (e.g., 97.5%, 98%, 98.5%, 99.0%, 99.5%, 99.6%, 99.7%, 99.8%,99.9%, or 100%) sequence identity with a region of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 thatincludes one or more variants described herein. The region of SEQ IDNO:1, 2, 3, 4, 5, or 6 is at least ten nucleotides in length (e.g., ten,15, 20, 50, 60, 70, 75, 100, 150 or more nucleotides in length). Forexample, a nucleic acid molecule can have at least 99% identity with aregion of SEQ ID NO:6 containing nucleotides −100 to −1, 1 to 100, 50 to150, 200 to 300, 400 to 500, or 700 to 800 relative to the adenine ofthe SULT1E1 translation initiation codon, where the nucleotide sequenceof SEQ ID NO:6 includes one or more of the variants described herein.For example, the nucleotide sequence of SEQ ID NO:6 can have, withrespect to the adenine of the SULT1E1 translation initiation site, anadenine at position −69, a thymine at position 64, a thymine at position95, a cytosine at position 237, a thymine at position 459, or an adenineat position 758, and combinations thereof.

In another embodiment, a nucleic acid molecule can have at least 99%identity with a region of SEQ ID NO:1 containing nucleotides −275 to−175 or −250 to −150 relative to the adenine of the SULT1E1 translationinitiation codon, nucleotides 1 to 100 relative to the guanine in thesplice donor site of intron 1, nucleotides −125 to −25 or −100 to −1relative to the guanine in the splice acceptor site of intron 1, ornucleotides 1 to 100 relative to the guanine in the splice donor site ofintron 2, where the nucleotide sequence of SEQ ID NO:1 includes one ormore of the variants described herein. For example, the nucleotidesequence of SEQ ID NO:1 can have an adenine at position −232 relative tothe adenine of the SULT1E1 translation initiation site (position 643 ofSEQ ID NO:1), a guanine at position −190 relative to the adenine of theSULT1E1 translation initiation site (position 685 of SEQ ID NO:1), aguanine at position 69 relative to the guanine of the splice donor siteof intron 1, a cytosine at position −73 relative to the guanine in thesplice acceptor site of intron 1, a thymine at position −20 relative tothe guanine in the splice acceptor site of intron 1, or a cytosine atposition 22 relative to the guanine in the splice donor site of intron2, and combinations thereof.

In another embodiment, a nucleic acid molecule can have at least 99%identity with a region of SEQ ID NO:2 containing nucleotides −175 to −75or −125 to −25 relative to the guanine in the splice acceptor site ofintron 3 or nucleotides 1 to 100 or 75 to 175 relative to the guanine inthe splice donor site of intron 4, where the nucleotide sequence of SEQID NO:2 includes one or more of the variants described herein. Forexample, the nucleotide sequence of SEQ ID NO:2 can have a guanine atnucleotide −137 or at nucleotide −80 relative to the guanine of thesplice acceptor site of intron 3 or a thymine at position 69 or 139relative to the guanine in the splice donor site of intron 4, andcombinations thereof.

In still another embodiment, a nucleic acid molecule can have at least99% identity with a region of SEQ ID NO:3 containing nucleotides −100 to−1 relative to the guanine in the splice acceptor site of intron 4,nucleotides 1 to 100 relative to the guanine in the splice donor site ofintron 5, nucleotides −100 to −1 relative to the guanine in the spliceacceptor site of intron 5, or nucleotides 1 to 100 relative to theguanine in the splice donor site of intron 6, where the nucleotidesequence of SEQ ID NO:3 includes one or more of the variants describedherein. For example, the nucleotide sequence of SEQ ID NO:3 can have aguanine at nucleotide −23 relative to the guanine in the splice acceptorsite of intron 4, a thymine at nucleotide 55 relative to the guanine inthe splice donor site of intron 5, a guanine at position −10 relative tothe guanine in the splice acceptor site of intron 5, or a deletion atposition 55 relative to the guanine in the splice donor site of intron6, and combinations thereof.

A nucleic acid molecule also can have at least 99% identity with aregion of SEQ ID NO:4 containing nucleotides −100 to −1 relative to theguanine in the splice acceptor site of intron 6, where the nucleotidesequence of SEQ ID NO:4 includes one or more of the variants describedherein. For example, the nucleotide sequence of SEQ ID NO:4 can have acytosine at nucleotide −39 relative to the guanine in the spliceacceptor site of intron 6. In another embodiment, a nucleic acidmolecule can have at least 99% identity with a region of SEQ ID NO:5containing nucleotides −175 to −125 or nucleotides −100 to −1 relativeto the guanine in the splice acceptor site of intron 7, where thenucleotide sequence of SEQ ID NO:5 includes one or more of the variantsdescribed herein. For example, the nucleotide sequence of SEQ ID NO:5can have a thymine at nucleotide −121 relative to the guanine in thesplice acceptor site of intron 7 or a guanine at nucleotide −63 relativeto the guanine in the splice acceptor site of intron 7, or combinationsthereof.

Percent sequence identity is calculated by determining the number ofmatched positions in aligned nucleic acid sequences, dividing the numberof matched positions by the total number of aligned nucleotides, andmultiplying by 100. A matched position refers to a position in whichidentical nucleotides occur at the same position in aligned nucleic acidsequences. Percent sequence identity also can be determined for anyamino acid sequence. To determine percent sequence identity, a targetnucleic acid or amino acid sequence is compared to the identifiednucleic acid or amino acid sequence using the BLAST 2 Sequences (B12seq)program from the stand-alone version of BLASTZ containing BLASTN version2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ canbe obtained from Fish & Richardson's web site (World Wide Web atfr.com/blast) or the U.S. government's National Center for BiotechnologyInformation web site. Instructions explaining how to use the B12seqprogram can be found in the readme file accompanying BLASTZ.

B12seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: −iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); −j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); −p is set toblastn; −o is set to any desired file name (e.g., C:\output.txt); −q isset to −1; −r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\B12seq −i c:\seq1.txt−j c:\seq2.txt −p blastn −o c:\output.txt −q −1 −r 2. If the targetsequence shares homology with any portion of the identified sequence,then the designated output file will present those regions of homologyas aligned sequences. If the target sequence does not share homologywith any portion of the identified sequence, then the designated outputfile will not present aligned sequences.

Once aligned, a length is determined by counting the number ofconsecutive nucleotides from the target sequence presented in alignmentwith sequence from the identified sequence starting with any matchedposition and ending with any other matched position. A matched positionis any position where an identical nucleotide is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides. Likewise, gaps presentedin the identified sequence are not counted since target sequencenucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length is determined by countingthe number of matched positions over that length and dividing thatnumber by the length followed by multiplying the resulting value by 100.For example, if (1) a 1000 nucleotide target sequence is compared to thesequence set forth in SEQ ID NO:6, (2) the B12seq program presents 900nucleotides from the target sequence aligned with a region of thesequence set forth in SEQ ID NO: 1 where the first and last nucleotidesof that 900 nucleotide region are matches, and (3) the number of matchesover those 900 aligned nucleotides is 850, then the 1000 nucleotidetarget sequence contains a length of 900 and a percent identity overthat length of 94 (i.e., 850)900×100=94).

It will be appreciated that different regions within a single nucleicacid target sequence that aligns with an identified sequence can eachhave their own percent identity. It is noted that the percent identityvalue is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13,and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18,and 78.19 are rounded up to 78.2. It also is noted that the length valuewill always be an integer.

Isolated nucleic acid molecules of the invention can be produced bystandard techniques, including, without limitation, common molecularcloning and chemical nucleic acid synthesis techniques. For example,polymerase chain reaction (PCR) techniques can be used to obtain anisolated nucleic acid containing a SULT1E1 nucleotide sequence variant.PCR refers to a procedure or technique in which target nucleic acids areenzymatically amplified. Sequence information from the ends of theregion of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified. PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. Primers are typically 14 to 40nucleotides in length, but can range from 10 nucleotides to hundreds ofnucleotides in length. General PCR techniques are described, for examplein PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler,Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complementaryDNA (cDNA) strands. Ligase chain reaction, strand displacementamplification, self-sustained sequence replication or nucleic acidsequence-based amplification also can be used to obtain isolated nucleicacids. See, for example, Lewis (1992) Genetic Engineering News 12:1;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA, 87:1874-1878; andWeiss (1991) Science 254:1292.

Isolated nucleic acids of the invention also can be chemicallysynthesized, either as a single nucleic acid molecule (e.g., usingautomated DNA synthesis in the 3′ to 5′ direction using phosphoramiditetechnology) or as a series of oligonucleotides. For example, one or morepairs of long oligonucleotides (e.g., >100 nucleotides) can besynthesized that contain the desired sequence, with each pair containinga short segment of complementarity (e.g., about 15 nucleotides) suchthat a duplex is formed when the oligonucleotide pair is annealed. DNApolymerase is used to extend the oligonucleotides, resulting in asingle, double-stranded nucleic acid molecule per oligonucleotide pair,which then can be ligated into a vector.

Isolated nucleic acids of the invention also can be obtained bymutagenesis. For example, the reference sequence depicted in FIG. 1 canbe mutated using standard techniques including oligonucleotide-directedmutagenesis and site-directed mutagenesis through PCR. See, ShortProtocols in Molecular Biology, Chapter 8, Green Publishing Associatesand John Wiley & Sons, ed. Ausubel et al., 1992. Examples of positionsthat can be modified include those described herein.

SULT1E1 Polypeptides

Isolated SULT1E1 polypeptides of the invention include an amino acidsequence variant relative to the reference SULT1E1 polypeptide (FIG. 2B;SEQ ID NO:7; GenBank Accession No. U08098). The term “isolated” withrespect to a SULT1E1 polypeptide refers to a polypeptide that has beenseparated from cellular components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60% (e.g.,70%, 80%, 90%, 95%, or 99%), by weight, free from proteins and naturallyoccurring organic molecules that are naturally associated with it. Ingeneral, an isolated polypeptide will yield a single major band on anon-reducing polyacrylamide gel.

SULT1E1 polypeptides of the invention include variants at one or more ofresidues 22, 32, and 253. In particular, a tyrosine residue can besubstituted at position 22, a valine residue at position 32, or ahistidine at position 253. SULT1E1 polypeptides may have more than oneamino acid substitution.

In some embodiments, the activity of SULT1E1 allozymes can be alteredrelative to the reference SULT1E1 polypeptide. As described herein,certain SULT1E1 allozymes have reduced activity (e.g., Asp22Tyr andAla32Val). In other embodiments, the activity of SULT1E1 allozymes(e.g., Pro253His) have activity that is similar to that of the referenceSULT1E1 polypeptide. The activity of SULT1E1 polypeptides can bemeasured as described by Foldes and Meek (Biochim. Biophys. Acta,327:365-374, 1973) or Hernandez et al. (Drug Metab. Disposit.20:413-422, 1992). Briefly, SULT1E1 activity can be assayed in vitrousing a sulfate acceptor substrate such as 17-β estradiol (E2, SigmaChemical Co., St. Louis, Mo.) and a donor sulfate molecule such as PAPS.In general, recombinant SULT1E1 polypeptides can be incubated at 37° C.with 0.05 μM of sulfate acceptor substrate and 0.4 μM labeled PAPS(e.g., ³⁵S-PAPS from New England Nuclear Life Science Products, Inc.,Boston Mass.). Reactions can be stopped by precipitating unreacted PAPSand SULT1E1 polypeptide (e.g., with barium hydroxide, barium acetate,and zinc sulfate). After centrifugation of the reaction mixture,radioactivity in the supernatant can be assessed. SULT1E1 activity canbe expressed as nmole of sulfate conjugated product formed per hour ofincubation. See Campbell et al. (1987) Biochem. Pharmacol. 36:1435-1446.

Other biochemical properties of allozymes, such as apparent K_(m)values, also can be altered relative to the reference SULT1E1. ApparentK_(m) values can be calculated, for example, by using the method ofWilkinson with a computer program written by Cleland. Wilkinson (1961)Biochem. J. 80:324-332 and Cleland (1963) Nature 198:463-365. Asdescribed herein, the apparent K_(m) values for PAPS varied more than3-fold among the allozymes tested (Asp22Tyr, Ala32Val, and Pro253His).

Isolated polypeptides of the invention can be obtained, for example, byextraction from a natural source (e.g., liver tissue), chemicalsynthesis, or by recombinant production in a host cell. To recombinantlyproduce SULT1E1 polypeptides, a nucleic acid sequence encoding asulfotransferase variant polypeptide can be ligated into an expressionvector and used to transform a bacterial or eukaryotic host cell (e.g.,insect, yeast, or mammalian cells). In general, nucleic acid constructsinclude a regulatory sequence operably linked to a sulfotransferasenucleic acid sequence. Regulatory sequences do not typically encode agene product, but instead affect the expression of the nucleic acidsequence. In addition, a construct can include a tag sequence designedto facilitate subsequent manipulations of the expressed nucleic acidsequence (e.g., purification, localization). Tag sequences, such asgreen fluorescent protein (GFP), glutathione S-transferase (GST), sixhistidine (His₆), c-myc, hemagglutinin, or Flag™ tag (Kodak) sequencesare typically expressed as a fusion with the expressed nucleic acidsequence. Such tags can be inserted anywhere within the polypeptideincluding at either the carboxyl or amino termini. The type andcombination of regulatory and tag sequences can vary with eachparticular host, cloning or expression system, and desired outcome. Inbacterial systems, a strain of Escherichia coli such as BL-21 can beused. Suitable E. coli vectors include the pGEX series of vectors thatproduce fusion proteins with glutathione S-transferase (GST).Transformed E. coli are typically grown exponentially, then stimulatedwith isopropylthiogalactopyranoside (IPTG) prior to harvesting. Ingeneral, such fusion proteins are soluble and can be purified easilyfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In eukaryotic host cells, a number of viral-based expression systems canbe utilized to express sulfotransferase variant polypeptides. A nucleicacid encoding a polypeptide of the invention can be cloned into, forexample, a baculoviral vector such as pBlueBac (Invitrogen, San Diego,Calif.) and then used to co-transfect insect cells such as Spodopterafrugiperda (Sf9) cells with wild type DNA from Autographa californicamulti-nuclear polyhedrosis virus (AcMNPV). Recombinant viruses producingpolypeptides of the invention can be identified by standard methodology.Alternatively, a nucleic acid encoding a polypeptide of the inventioncan be introduced into a SV40, retroviral, or vaccinia based viralvector and used to infect suitable host cells.

Mammalian cell lines that stably express sulfotransferase variantpolypeptides can be produced by using expression vectors with theappropriate control elements and a selectable marker. For example, theeukaryotic expression vector pCR3.1 (Invitrogen, San Diego, Calif.) andp91023(B) are suitable for expression of sulfotransferase variantpolypeptides in mammalian cells such as Chinese hamster ovary (CHO)cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells,BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC).Following introduction of the expression vector by electroporation,lipofection, calcium phosphate or calcium chloride co-precipitation,DEAE dextran, or other suitable method, stable cell lines are selected,e.g., by antibiotic resistance to G418, kanamycin, or hygromycin.Alternatively, amplified sequences can be ligated into a mammalianexpression vector such as pcDNA3 (Invitrogen, San Diego, Calif.) andthen transcribed and translated in vitro using wheat germ extract orrabbit reticulocyte lysate.

SULT1E1 variant polypeptides can be purified by known chromatographicmethods including DEAE ion exchange, gel filtration, and hydroxyapatitechromatography. See, Van Loon and Weinshilboum (1990) Drug Metab.Dispos. 18:632-638; and Van Loon et al. (1992) Biochem. Pharmacol.44:775-785. SULT1E1 polypeptides can be “engineered” to contain an aminoacid sequence that allows the polypeptide to be captured onto anaffinity matrix. For example, a tag such as c-myc, hemagglutinin,polyhistidine, or Flag™ tag (Kodak) can be used to aid polypeptidepurification. Such tags can be inserted anywhere within the polypeptideincluding at either the carboxyl or amino termini. Other fusions thatcould be useful include enzymes that aid in the detection of thepolypeptide, such as alkaline phosphatase. Immunoaffinity chromatographyalso can be used to purify SULT1E1 polypeptides.

Non-Human Mammals

The invention features non-human mammals that include SULT1E1 nucleicacids of the invention, as well as progeny and cells of such non-humanmammals. Non-human mammals include, for example, rodents such as rats,guinea pigs, and mice, and farm animals such as pigs, sheep, goats,horses and cattle. Non-human mammals of the invention can express aSULT1E1 variant nucleic acid in addition to an endogenous SULT1E1 (e.g.,a transgenic non-human that includes a SULT1E1 nucleic acid randomlyintegrated into the genome of the non-human mammal). Alternatively, anendogenous SULT1E1 nucleic acid can be replaced by a SULT1E1 variantnucleic acid of the invention through homologous recombination. SeeShastry (1998) Mol. Cell. Biochem. 181:163-179, for a review of genetargeting technology.

In one embodiment, non-human mammals are produced that lack anendogenous SULT1E1 nucleic acid (i.e., a knockout), then a SULT1E1variant nucleic acid of the invention is introduced into the knockoutnon-human mammal. Nucleic acid constructs used for producing knockoutnon-human mammals can include a nucleic acid sequence encoding aselectable marker, which is generally used to interrupt the targetedexon site by homologous recombination. Typically, the selectable markeris flanked by sequences homologous to the sequences flanking the desiredinsertion site. It is not necessary for the flanking sequences to beimmediately adjacent to the desired insertion site. Suitable markers forpositive drug selection include, for example, the aminoglycoside 3Nphosphotransferase gene that imparts resistance to geneticin (G418, anaminoglycoside antibiotic), and other antibiotic resistance markers,such as the hygromycin-B-phosphotransferase gene that imparts hygromycinresistance. Other selection systems include negative-selection markerssuch as the thymidine kinase (TK) gene from herpes simplex virus.Constructs utilizing both positive and negative drug selection also canbe used. For example, a construct can contain the aminoglycosidephosphotransferase gene and the TK gene. In this system, cells areselected that are resistant to G418 and sensitive to gancyclovir.

To create non-human mammals having a particular gene inactivated in allcells, it is necessary to introduce a knockout construct into the germcells (sperm or eggs, i.e., the “germ line”) of the desired species.Genes or other DNA sequences can be introduced into the pronuclei offertilized eggs by microinjection. Following pronuclear fusion, thedeveloping embryo may carry the introduced gene in all its somatic andgerm cells since the zygote is the mitotic progenitor of all cells inthe embryo. Since targeted insertion of a knockout construct is arelatively rare event, it is desirable to generate and screen a largenumber of animals when employing such an approach. Because of this, itcan be advantageous to work with the large cell populations andselection criteria that are characteristic of cultured cell systems.However, for production of knockout animals from an initial populationof cultured cells, it is necessary that a cultured cell containing thedesired knockout construct be capable of generating a whole animal. Thisis generally accomplished by placing the cell into a developing embryoenvironment of some sort.

Cells capable of giving rise to at least several differentiated celltypes are “pluripotent.” Pluripotent cells capable of giving rise to allcell types of an embryo, including germ cells, are hereinafter termed“totipotent” cells. Totipotent murine cell lines (embryonic stem, or“ES” cells) have been isolated by culture of cells derived from veryyoung embryos (blastocysts). Such cells are capable, upon incorporationinto an embryo, of differentiating into all cell types, including germcells, and can be employed to generate animals lacking an endogenousSULT1E1 nucleic acid. That is, cultured ES cells can be transformed witha knockout construct and cells selected in which the SULT1E1 gene isinactivated.

Nucleic acid constructs can be introduced into ES cells by, for example,electroporation or other standard technique. Selected cells can bescreened for gene targeting events. For example, the polymerase chainreaction (PCR) can be used to confirm the presence of the transgene.

The ES cells further can be characterized to determine the number oftargeting events. For example, genomic DNA can be harvested from EScells and used for Southern analysis. See, for example, Section9.37-9.52 of Sambrook et al., Molecular Cloning, A Laboratory Manual,second edition, Cold Spring Harbor Press, Plainview, N.Y., 1989.

To generate a knockout animal, ES cells having at least one inactivatedSULT1E1 allele are incorporated into a developing embryo. This can beaccomplished through injection into the blastocyst cavity of a murineblastocyst-stage embryo, by injection into a morula-stage embryo, byco-culture of ES cells with a morula-stage embryo, or through fusion ofthe ES cell with an enucleated zygote. The resulting embryo is raised tosexual maturity and bred in order to obtain animals, whose cells(including germ cells) carry the inactivated SULT1E1 allele. If theoriginal ES cell was heterozygous for the inactivated SULT1E1 allele,several of these animals can be bred with each other in order togenerate animals homozygous for the inactivated allele.

Alternatively, direct microinjection of DNA into eggs can be used toavoid the manipulations required to turn a cultured cell into an animal.Fertilized eggs are “totipotent,” i.e., capable of developing into anadult without further substantive manipulation other than implantationinto a surrogate mother. To enhance the probability of homologousrecombination when eggs are directly injected with knockout constructs,it is useful to incorporate at least about 8 kb of homologous DNA intothe targeting construct. In addition, it is also useful to prepare theknockout constructs from isogenic DNA.

Embryos derived from microinjected eggs can be screened for homologousrecombination events in several ways. For example, if the SULT1E1 geneis interrupted by a coding region that produces a detectable (e.g.,fluorescent) gene product, then the injected eggs are cultured to theblastocyst stage and analyzed for presence of the indicator polypeptide.Embryos with fluorescing cells, for example, are then implanted into asurrogate mother and allowed to develop to term. Alternatively, injectedeggs are allowed to develop and DNA from the resulting pups analyzed byPCR or RT-PCR for evidence of homologous recombination.

Nuclear transplantation also can be used to generate non-human mammalsof the invention. For example, fetal fibroblasts can be geneticallymodified such that they contain an inactivated SULT1E1 gene, and thenfused with enucleated oocytes. After activation of the oocytes, the eggsare cultured to the blastocyst stage, and implanted into a recipient.See Cibelli et al. (1998) Science 280:1256-1258. Adult somatic cells,including, for example, cumulus cells and mammary cells, can be used toproduce animals such as mice and sheep, respectively. See, for example,Wakayama et al. (1998) Nature 394:369-374; and Wilmut et al. (1997)Nature 385:810-813. Nuclei can be removed from genetically modifiedadult somatic cells, and transplanted into enucleated oocytes. Afteractivation, the eggs can be cultured to the 2-8 cell stage, or to theblastocyst stage, and implanted into a suitable recipient. See, Wakayamaet al. (1998) supra.

Non-human mammals of the invention such as mice can be used to screen,for example, toxicity of compounds that are substrates for SULT1E1,drugs that alter SULT1E1 activity, or for carcinogenesis. For example,SULT1E1 activity or toxicity can be assessed in a first group of suchnon-human mammals in the presence of a compound, and compared withSULT1E1 activity or toxicity in a corresponding control group in theabsence of the compound. As used herein, suitable compounds includebiological macromolecules such as an oligonucleotide (RNA or DNA), or apolypeptide of any length, a chemical compound, a mixture of chemicalcompounds, or an extract isolated from bacterial, plant, fungal, oranimal matter. The concentration of compound to be tested depends on thetype of compound and in vitro test data.

Non-human mammals can be exposed to test compounds by any route ofadministration, including enterally and parenterally. For example, thecompound can be administered parenterally through inhalation, or byintranasal, intravascular, intramuscular, or subcutaneousadministration. Enteral routes include sublingual and oraladministration. Compounds can be prepared for parenteral administrationin the form of liquid solutions or suspensions; for oral administrationin the form of tablets or capsules; or for intranasal administration inthe form of powders, nasal drops, or aerosols. Compounds can be preparedfor other routes of administration using standard techniques. Testcompounds can be mixed with non-toxic excipients or carriers beforeadministration. Inhalation formulations can include aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholate,or deoxycholate. Other formulations may contain sterile water or saline,or polyalkylene glycols such as polyethylene glycol.

Detecting Sulfotransferase Sequence Variants

Sulfotransferase nucleotide sequence variants can be detected by, forexample, sequencing exons, introns, 5′-untranslated sequences, or3′-untranslated sequences, by performing allele-specific hybridization,allele-specific restriction digests, mutation specific polymerase chainreactions (MSPCR), by single-stranded conformational polymorphism (SSCP)detection (Schafer et al. (1995) Nat. Biotechnol. 15:33-39), denaturinghigh performance liquid chromatography (DHPLC; Underhill et al. (1997)Genome Res. 7:996-1005), infrared matrix-assisted laserdesorption/ionization (IR-MALDI) mass spectrometry (WO 99/57318), andcombinations of such methods. Genomic DNA generally is used in theanalysis of sulfotransferase nucleotide sequence variants. Genomic DNAtypically is extracted from a biological sample such as a peripheralblood sample, but can be extracted from other biological samples,including tissues (e.g., mucosal scrapings of the lining of the mouth orfrom renal or hepatic tissue). Routine methods can be used to extractgenomic DNA from a blood or tissue sample, including, for example,phenol extraction. Alternatively, genomic DNA can be extracted with kitssuch as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.), Wizard®Genomic DNA purification kit (Promega, Madison, Wis.) and the A.S.A.P.™Genomic DNA isolation kit (Boehringer Mannheim, Indianapolis, Ind.).

Typically, an amplification step is performed before proceeding with thedetection method. For example, exons or introns of the sulfotransferasegene can be amplified then directly sequenced. Dye primer sequencing canbe used to increase the accuracy of detecting heterozygous samples.

Allele specific hybridization also can be used to detect sequencevariants, including complete haplotypes of a mammal. See, Stoneking etal. (1991) Am. J. Hum. Genet. 48:370-382; and Prince et al. (2001)GenomeRes. 11:152-162. In practice, samples of DNA or RNA from one or moremammals can be amplified using pairs of primers and the resultingamplification products can be immobilized on a substrate (e.g., indiscrete regions). Hybridization conditions are selected such that anucleic acid probe can specifically bind to the sequence of interest,e.g., the variant nucleic acid sequence. Such hybridizations typicallyare performed under high stringency as some sequence variants includeonly a single nucleotide difference. High stringency conditions caninclude the use of low ionic strength solutions and high temperaturesfor washing. For example, nucleic acid molecules can be hybridized at42° C. in 2×SSC (0.3M NaCl/0.03 M sodium citrate/0.1% sodium dodecylsulfate (SDS) and washed in 0.1×SSC (0.015M NaCl/0.0015 M sodiumcitrate), 0.1% SDS at 65° C. Hybridization conditions can be adjusted toaccount for unique features of the nucleic acid molecule, includinglength and sequence composition. Probes can be labeled (e.g.,fluorescently) to facilitate detection. In some embodiments, one of theprimers used in the amplification reaction is biotinylated (e.g., 5′-endof reverse primer) and the resulting biotinylated amplification productis immobilized on an avidin or streptavidin coated substrate.Allele-specific restriction digests can be performed in the followingmanner. For nucleotide sequence variants that introduce a restrictionsite, restriction digest with the particular restriction enzyme candifferentiate the alleles. For SULT1E1 sequence variants that do notalter a common restriction site, mutagenic primers can be designed thatintroduce a restriction site when the variant allele is present or whenthe wild type allele is present. A portion of SULT1E1 nucleic acid canbe amplified using the mutagenic primer and a wild type primer, followedby digest with the appropriate restriction endonuclease.

Certain variants, such as insertions or deletions of one or morenucleotides, change the size of the DNA fragment encompassing thevariant. The nucleotide insertion or deletion can be assessed byamplifying the region encompassing the variant. The size of theamplified products can be determined by comparison with size standards.For example, a region of SULT1E1 containing the deletion in intron 6 ofcan be amplified using a primer set from either side of the variant. Oneof the primers typically is labeled with, for example, a fluorescentmoiety, to facilitate sizing. The amplified products can beelectrophoresed through acrylamide gels with a set of size standardsthat are labeled with a fluorescent moiety that differs from the primer.

PCR conditions and primers can be developed that amplify a product onlywhen the variant allele is present or only when the wild type allele ispresent (MSPCR or allele-specific PCR). For example, patient DNA and acontrol can be amplified separately using either a wild type primer or aprimer specific for the variant allele. Each set of reactions is thenexamined for the presence of amplification products using standardmethods to visualize the DNA. For example, the reactions can beelectrophoresed through an agarose gel and the DNA visualized bystaining with ethidium bromide or other DNA intercalating dye. In DNAsamples from heterozygous patients, reaction products would be detectedin each reaction. Patient samples containing solely the wild type allelewould have amplification products only in the reaction using the wildtype primer. Similarly, patient samples containing solely the variantallele would have amplification products only in the reaction using thevariant primer. Allele-specific PCR also can be performed usingallele-specific primers that introduce priming sites for two universalenergy-transfer-labeled primers (e.g., one primer labeled with a greendye such as fluorescein and one primer labeled with a red dye such assulforhodamine). Amplification products can be analyzed for green andred fluorescence in a plate reader. See Myakishev et al. (2001) Genome11:163-169.

Mismatch cleavage methods also can be used to detect differing sequencesby PCR amplification, followed by hybridization with the wild typesequence and cleavage at points of mismatch. Chemical reagents such ascarbodiimide or hydroxylamine and osmium tetroxide can be used to modifymismatched nucleotides to facilitate cleavage. Alternatively, SULT1E1polypeptide variants can be detected using antibodies that have specificbinding affinity for SULT1E1 allozymes. Variant SULT1E1 polypeptides canbe produced in a number of ways, including recombinantly, as discussedabove. Host animals such as rabbits, chickens, mice, guinea pigs, andrats can be immunized by injection of a SULT1E1 variant polypeptide.Adjuvants that can be used to increase the immunological response dependon the host species and include Freund's adjuvant (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Polyclonalantibodies are heterogeneous populations of antibody molecules that arecontained in the sera of the immunized animals. Monoclonal antibodies,which are homogeneous populations of antibodies to a particular antigen,can be prepared using a sulfotransferase variant polypeptide andstandard hybridoma technology. In particular, monoclonal antibodies canbe obtained by any technique that provides for the production ofantibody molecules by continuous cell lines in culture such as describedby Kohler et al. (1975) Nature 256:495, the human B-cell hybridomatechnique (Kosbor et al. (1983) Immunology Today 4:72; Cole et al.(1983) Proc. Natl. Acad. Sci. USA 80:2026), and the EBV-hybridomatechnique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96. Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the monoclonal antibodies of theinvention can be cultivated in vitro and in vivo.

Antibody fragments that have specific binding affinity for a SULT1E1variant polypeptide can be generated by known techniques. Such fragmentsinclude, but are not limited to, F(ab′)2 fragments that can be producedby pepsin digestion of the antibody molecule, and Fab fragments that canbe generated by reducing the disulfide bridges of F(ab′)2 fragments.Alternatively, Fab expression libraries can be constructed. See, forexample, Huse et al. (1989) Science 246:1275. Once produced, antibodiesor fragments thereof are tested for recognition of sulfotransferasevariant polypeptides by standard immunoassay methods including ELISAtechniques, radioimmunoassays, and Western blotting. See, ShortProtocols in Molecular Biology, Chapter 11, Green Publishing Associatesand John Wiley & Sons, Edited by Ausubel et al., 1992.

Methods

As a result of the present invention, it is now possible to determinethe sulfonator status of a mammal (e.g., a human subject) as well as todetermine if particular SNPs are linked to a particular disease orclinical condition. In some embodiments, for example, it is possible todetermine whether a mammal is predisposed (i.e., has a relative greaterrisk) to a disease such as cancer (e.g., an estrogen responsive cancer).“Sulfonator status” refers to the ability of a mammal to transfer asulfate group to a substrate. The presence of SULT1E1 allozymes withreduced activity may indicate a relatively increased risk fordevelopment of estrogen responsive cancers such as breast cancer orovarian cancer. Additional risk factors including, for example, familyhistory and other genetic factors can be considered when determiningrisk.

Sulfonator status or predisposition to cancer can be determined based onthe presence or absence of a single SULT1E1 sequence variant or based ona variant profile. “Variant profile” refers to the presence or absenceof a plurality (i.e., two or more sequence variants) of SULT1E1nucleotide sequence variants or SULT amino acid sequence variants. Forexample, a variant profile can include the complete SULT1E1 haplotype ofthe mammal or can include the presence or absence of a set of commonnon-synonymous SNPs (i.e., single nucleotide substitutions that alterthe amino acid sequence of a SULT1E1 polypeptide). Non-limiting examplesof SULT1E1 haplotypes (haplotypes *1A-*1L, *2, *3, and *4) are found inTable 4. In one embodiment, the variant profile includes detecting thepresence or absence of two or more non-synonymous SNPs (e.g., 2, 3, 4,5, 6, or 7 non-synonymous SNPs and combinations thereof) describedabove. There may be ethnic-specific pharmacogenetic variation, ascertain of the nucleotide and amino acid sequence variants describedherein were detected solely in a particular ethnic group (i.e., a groupof African-American subjects or a group of Caucasian subjects). Inaddition, the variant profile can include detecting the presence orabsence of any type of SULT1E1 SNP together with any other SULT1E1 SNP(i.e., a polymorphism pair or groups of polymorphism pairs). Suchpolymorphism pairs include, without limitation, those pairs described inTable 3. Furthermore, the variant profile can include detecting thepresence or absence of any SULT1E1 SNP together with any SNP fromanother SULT nucleic acid.

Articles of Manufacture

The invention provides articles of manufacture that contain populationsof isolated SULT1E1 nucleic acid molecules or SULT1E1 polypeptidesimmobilized on a substrate. Suitable substrates provide a base for theimmobilization of the nucleic acids or polypeptides, and in someembodiments, allow immobilization of nucleic acids or polypeptides intodiscrete regions. In embodiments in which the substrate includes aplurality of discrete regions, different populations of isolated nucleicacids or polypeptides can be immobilized in each discrete region. Thus,each discrete region of the substrate can include a different SULT1E1nucleic acid or SULT1E1 polypeptide sequence variant. Such articles ofmanufacture can include two or more SULT1E1 sequence variants, or caninclude all of the sequence variants known for SULT1E1. Furthermore,nucleic acid molecules containing sequence variants for othersulfotransferases, such as SULT1A1, SULT1A2, or SULT1A3, can be includedon the substrate. See, WO 99/64630 and WO 00/20605 for a description ofother SULT1A1, SULT1A2, and SULT1A3 sequence variants.

Suitable substrates can be of any shape or form and can be constructedfrom, for example, glass, silicon, metal, plastic, cellulose or acomposite. For example, a suitable substrate can include a multiwellplate or membrane, a glass slide, a chip, or polystyrene or magneticbeads. Nucleic acid molecules or polypeptides can be synthesized insitu, immobilized directly on the substrate, or immobilized via alinker, including by covalent, ionic, or physical linkage. Linkers forimmobilizing nucleic acids and polypeptides, including reversible orcleavable linkers, are known in the art. See, for example, U.S. Pat. No.5,451,683 and WO98/20019. Immobilized nucleic acid molecules aretypically about 20 nucleotides in length, but can vary from about 10nucleotides to about 1000 nucleotides in length.

In practice, a sample of DNA or RNA from a subject can be amplified, theamplification product hybridized to an article of manufacture containingpopulations of isolated nucleic acid molecules in discrete regions, andhybridization can be detected. Typically, the amplified product islabeled to facilitate detection of hybridization. See, for example,Hacia et al. (1996) Nature Genet. 14:441-447; and U.S. Pat. Nos.5,770,722 and 5,733,729.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 PCR Amplification and DNA Sequencing

60 African American and 60 Caucasian genomic DNA samples were obtainedfrom the Coriell Cell Repository (Coriell Institute for MedicalResearch, Camden, N.J.). Specifically, 60 genomic DNA samples each fromthe two 100 item sample sets HD100AA and HD100CAU were used to astemplates in PCR with SULT1E1-specific primers. All DNA samples had beenanonymized by the National Institutes of Health prior to their depositin the Coriell Cell Repository. To make it possible to sequence SULT1E1,the eight exons in the gene were amplified from each of the 120 DNAsamples by use of PCR. Specifically, PCR primers were designed thatflanked the exons and that would produce amplification products 400-500base pairs in length. Therefore, eight separate amplifications wereperformed for each DNA sample.

Amplification reactions were performed with AmpliTaq Gold DNA polymerase(Perkin Elmer, Foster City, Calif.) with a “hot start” to help ensureamplification specificity. Each 50 μl reaction mixture contained 2.5units of DNA polymerase, 5 μl of a 10-fold diluted DNA sample (160-190ng DNA), 12.5 units of each primer (7 pmol for exon 7), 0.05 mM dNTPs(Boehringer Mannheim, Indianapolis, Ind.) and 5 μl of 10×PCR buffercontaining 15 mM MgCl₂ (Perkin Elmer). PCR cycling parameters involved a12 min “hot start” at 94° C., followed by 35 cycles (40 cycles for exon7) of 94° C. for 30 s, 55° C. (68° C. for the exon 7) for 30 s and 45 sat 72° C.—with a final 10 min extension at 72° C.

DNA sequencing was used to identify heterozygous bases. Sequencing wasperformed in the Mayo Clinic Molecular Biology Core Facility with anApplied Biosystems Model 377 DNA sequencers and BigDye™ (Perkin Elmer,Foster City, Calif.) dye primer sequencing chemistry. In all cases, bothDNA strands were sequenced.

Primers used in sequencing were tagged at the 5′-ends with M13 sequencetags. Locations of primers were chosen to avoid repetitive sequence andto ensure amplification specificity. The sequences and locations of eachprimer within the gene are listed in Table 1. All forward primerscontained the M13 forward sequence (underlined), and all reverse primerscontained the M13 reverse sequence (underlined) to make it possible touse dye primer DNA sequencing chemistry. “F” represents forward; “R,”reverse; “U,” upstream; “D,” downstream; “I,” intron; and “FR,” flankingregion. The numbering scheme for primers located in exons and the 5′-FRis based on the cDNA sequence, with the “A” at the translationinitiation codon designated as (+1). Positions 5′ and 3′ to thatlocation were assigned negative or positive numbers, respectively.Intron based primers were numbered on the basis of nucleotide distancefrom splice junctions, with (+1) as the first nucleotide at the 5′-end,and (−1) as the first nucleotide at the 3′-end of the intron.

The PolyPhred 3.0 and Consed 8.0 programs were used to analyze the DNAsequence chromatograms for polymorphic sites. The University ofWisconsin GCG software package, Version 10, also was used to analyzenucleotide sequence.

TABLE 1PCR primers used for SULT1E1 resequencing and site-directed mutagenesisPrimer SEQ ID Primer Name LocationPrimer Sequence Gene Specific Primer-3′ NO: Gene resequencing primersUF(−289)M13 5′-FRTGTAAAACGACGGCCAGTGCAGGATATTTCTACATCTCCATGAATGAACATGACT 8 I1R(147)Intron 1CAGGAAACAGCTATGACCGCTTCACATCATTAATTAACTAAAGTATCAAATCAAGACTTTGGTC 9I1F(−170) Intron 1TGTAAAACGACGGCCAGTCTCTCTAGTTACCCAAACTATTTGATATGCAACTTTGC 10 I2R(145)Intron 2 CAGGAAACAGCTATGACCGAGCTACCTTTTCTATGTCCATATCCAAACTACCG 11I2F(−151) Intron 2TGTAAAACGACGGCCAGTATAGAAAATATTTCCTGAGTCTGTGGCTATTCAGACACC 12 I3R(122)Intron 3 CAGGAAACAGCTATGACCGCTGTCTTATGTAGAAGACCTGATACTAATTGCCATTC 13I3F(−196) Intron 3TGTAAAACGACGGCCAGTTAGGCATGCAATGCATAATAATTACACCATGGGGAATG 14 I4R(185)Intron 4 CAGGAAACAGCTATGACCTGGCAAAAGACAGAGTTGGAATTAAAATATAGACTCTCTGAC 15I4F(−183) Intron 4 TGTAAAACGACGGCCAGTAAACCACTGTCACCTCAGGTTATTGAAGATGTCTT16 I5R(134) Intron 5 CAGGAAACAGCTATGACCATGCTTGCTCTTAAACCTCCAGGCCCCTTTAGA17 I5F(−171) Intron 5 TGTAAAACGACGGCCAGTCATGCTTTGCCTCTCTTGCTGGAGAGAACCT18 I6R(167) Intron 6CAGGAAACAGCTATGACCGCTTCAAATCTATGCTAAAGTATCTGTATTATTTTGGTCCTTTCC 19I6(−160) Intron 6TGTAAAACGACGGCCAGTCACAGCTTTTATAAAATTCCCCCAATTAGATTTCTCATTAGAAATC 20I7R(132) Intron 7CAGGAAACAGCTATGACCTCAAATATGAAAGACTGCTGAAGAAAACTTAAGCTGGGTT 21 I7F(−170)Intron 7 TGTAAAACGACGGCCAGTCATCTTTGTAAGCCCCCAAAAGTATATCATTAAAGGTATAC 22DR83 3′-FRCAGGAAACAGCTATGACCAGTTAAACAAAAATTTAAAAAGAAAATGTCAACATAATCCATGA 23Primers for site-directed mutagenesis F(−12) Exon 1TGTAAAACGACGGCCAGTCAGTGTACCACAATGAATTCTG 24 R890 Exon 7CAGGAAACAGCTATGACCCCTTCTTAGATCTCAGTTCGAA 25 F48 Exon 2TGTAAAACGACGGCCAGTGATTCTAATGTATAAA

ATTTTGTCAAATATTG 26 R80 Exon 2 CAGGAAACAGCTATGACCCAATATTTGACAAAAT

TTTATACATTAGAATC 27 F81 Exon 2 TGTAAAACGACGGCCAGTGGATAATGTGGAAG

GTTCCAGGCAAGAC 28 R109 Exon 2 CAGGAAACAGCTATGACCGTCTTGCCTGGAAC

CTTCCACATTATCC 29 F743 Exon 7 TGTAAAACGACGGCCAGTCCAGAAATTGTCGC

CTTCATGAGAAAGG 30 R772 Exon 7 CAGGAAACAGCTATGACCCCTTTCTCATGAAG

GCGACAATTTCTGG 31

Example 2 SULT1E1 Polymorphisms

The eight separate SULT1E1 PCR amplifications performed for each of the120 individual human genomic DNA samples studied generated approximately729,120 base pairs of sequence. The sequences were analyzed by use ofthe PolyPhred software.

All of the sequences analyzed were sequenced on both strands, making itpossible to use data from the opposite strand to verify polymorphismcalls. All sequences were compared to the SULT1E1 gene sequences ofGenBank accession numbers U08098 and U20514-U20521.

Sequencing of the 5′- and 3′-untranslated sequences, exons, and intronsof the SULT1E1 nucleic acid revealed 23 variations (Table 2); fifteenwere found in introns, six were found in exons, and two were found inthe 5′-flanking region. Polymorphisms in exons, untranslated regions(UTR), and flanking regions (FR) are numbered relative to the adenine inthe SULT1E1 translation initiation codon (ATG, adenine is +1).Polymorphisms in introns are numbered separately, either as positivenumbers relative to the guanine in the splice donor site (GT, guanine is+1), or as negative numbers relative to the guanine in the spliceacceptor site (AG, guanine is −1). Asterisks indicate insertions ordeletions. The average number of polymorphisms present both in the geneoverall and within the ORF was 7.6 per kilobase sequenced.

The lowest allele frequency that was possible to detect was 0.8% because60 DNA samples (120 alleles) were used. Those frequencies also arelisted in Table 2. For African-Americans, thirteen of the eighteenpolymorphisms had allele frequencies greater than 1% and, as a result,may be considered “common” in the African-American population sample.For Caucasian-Americans, eight of the thirteen polymorphisms had allelefrequencies greater than 1% and may be considered “common” in theCaucasian-American population sample. Four of the polymorphisms observedin each of the African-American and Caucasian-American populations hadallele frequencies greater than 10%. Six polymorphisms that wereconsidered common in the African-American population (I1(−120),I3(−137), I4(139), E5(459), I5(55), and I7(−63)) were not detected inthe Caucasian population, while one common polymorphism observed in theCaucasian population, I6(−39), was not detected in the African-Americanpopulation.

TABLE 2 Human SULT1E1 polymorphisms and frequencies Variant Sequence WTFrequency Polymorphism Location Sequence Amino Acid African- Caucasian-Position In Gene Nucleotide Nucleotide Change American American −2325′-FR G A 0.008 0.0 −190 5′-FR C G 0.008 0.0 −64 Exon 1 G A 0.2 0.067 69Intron 1 A G 0.225 0.492 −73 Intron 1 G C 0.3 0.383 −20 Intron 1 A T0.092 0.0 64 Exon 2 G T Asp22Tyr 0.008 0.0 95 Exon 2 C T Ala32Val 0.00.008 22 Intron 2 T C 0.008 0.008 237 Exon 3 T C 0.0 0.008 −137 Intron 3T G 0.017 0.0 −80 Intron 3 A G 0.017 0.033 69 Intron 4 A T 0.0 0.008 139Intron 4 A T 0.017 0.0 −23 Intron 4 A G 0.008 0.0 459 Exon 5 C T 0.0170.0 55 Intron 5 C T 0.017 0.0 −10 Intron 5 C G 0.083 0.117 55 Intron 6 GG Del 0.017 0.033 −39 Intron 6 T C 0.0 0.017 758 Exon 7 C A Pro253His0.0 0.008 −121 Intron 7 C T 0.333 0.108 −63 Intron 7 T G 0.017 0.0

Five SNPs were observed in the SULT1E1 coding region and one SNP(E1(−64)) in the UTR (Table 2 and FIG. 1). Two of the coding region SNPswere synonymous, i.e., they did not give rise to changes in amino acidsequence. The synonymous SNPs included E3(237) and E5(459). Three werenon-synonymous, i.e., they gave rise to amino acid substitutions. Thesesubstitutions, at E2(64), E2(95), and E7(758), resulted in threedifferent single variant SULT1E1 allozymes. One of the non-synonymouscSNPs, E2(64), was observed only in African-Americans. The other two,E2(95) and E7(758), were observed only in Caucasian-Americans. Inaddition to SNPs, one deletion event was observed in intron 6 of SULT1E1(Table 2 and FIG. 1).

Example 3 Linkage Disequilibrium and Haplotype Analysis

Linkage disequilibrium analysis was performed after all of the DNAsamples had been genotyped at each of the 23 polymorphic sites.Polymorphisms with p<0.05 were chosen for inclusion in this analysis,since there was inadequate statistical power for the analysis of lesscommon polymorphisms. All possible pairwise combinations of thesepolymorphisms were tested for linkage disequilibrium using the EHprogram developed by Terwilliger and Ott (1994) Handbook of HumanGenetic Linkage, The Johns Hopkins University Press, Baltimore, pp.188-193. The output of this program was used to calculate d′ values, amethod for reporting linkage data that is independent of sample size(see Table 3).

The genotype data also were used for haplotype analysis. In this case,unambiguous haplotype assignment could be made for samples thatcontained no more than one heterozygous locus. Haplotypes for some ofthe remaining alleles were inferred from the genotype data as well asfrom EM probabilities (see Table 4; Long et al. (1995) Am J Hum Genet.56:799-810; and Excoffier et al. (1995) Mol Biol Evol 12:921-927).

The twelve unambiguous haplotypes (those labeled *1) observed in the 120resequenced DNA samples are listed. Nucleotides within each of thealleles that differed from the SULT1E1 consensus sequence (*1C) areshown in lowercase bold type. Initial designations of haplotypes weremade on the basis of the encoded amino acid sequence, with the wild typesequence being designated *1. “Letter” designations were then addedbased on descending allele frequencies, starting with haplotypes presentin both ethnic groups and then making assignments based on haplotypesobserved in samples from African-American subjects. Although haplotypescould not be determined unequivocally, the Asp22Tyr variant is listed as*2, the Ala32Val variant as *3, and the Pro253His variant as *4.

TABLE 3 SULT1E1 linkage disequilibrium analysis African-AmericanCaucasian-American Polymorphism Pair d′ Value p-Value d′ Value p-Value−232 I3 (−80) 1 0.00694  — — −232 459 1 0.006494 — — −232 I6 (55) 10.006494 — — −232 I7 (−63) 1 0.006494 — —  −64 I1 (−73) −1 0.003557 — — −64 I7 (−121) 0.93 0     0.85 4e⁻⁰⁶ I1 (69)   I1 (−73) — — 0.65 1e⁻⁰⁶I1 (69)   I5 (−10) 1   3e⁻⁰⁶ 1 0.000506 I1 (−73) I7 (−121) −1 3.6e⁻⁰⁵ —— I3 (−80) I6 (55) 1 2.8e⁻⁰⁵ 1 0 I3 (−80) I6 (−39) — — 1 0.000544 I4(−23) I5 (55) 1 0.006494 — —  459 I7 (−63) 1 2.8e⁻⁰⁵ — — I6 (55)  I6(−39) — — 1 0.000544

TABLE 4 SULT1E1 haplotype analysis Nucleotide Position Frequency AlleleAfrican- Caucasian- Exon 1 Intron 1 Exon 2 Intron 4 Intron 5 Exon 7Intron 7 Designation American American −64 69 −73 −20 64 95 69 55 −10758 −121 *1A 0.250 0.071 G A c A G C A C C C C *1B 0.190 0.040 a A G A GC A C C C t *1C 0.106 0.326 G A G A G C A C C C C *1D 0.103 0.045 G A GA G C A C C C t *1E 0.067 0.102 G g G A G C A C g C C *1F 0.064 — G A Gt G C A C C C C *1G 0.050 0.276 G g c A G C A C C C C *1H 0.040 — G g GA G C A C C C t *1I 0.010 — a A G A G C A C C C C *1J 0.008 — G A G A GC A t C C C *1K — 0.045 G g G A G C A C C C C *1L — 0.009 G A G A G C tC C C C *2 0.008 — G A G A t C A C C C C *3 — 0.008 G A G A G t A C C CC *4 — 0.008 G A G A G C A C C a C

Example 4 SULT1E1 Expression

Four different SULT1E1 expression constructs were generated using thepCR3.1 expression vector (Invitrogen, Carlsbad, Calif.). Threeconstructs were designed to express the three variant SULT1E1polypeptides, while one construct was designed to express the wild typeSULT1E1. All variant SULT1E1 cDNA sequences used to make the expressionconstructs were created by site directed mutagenesis using theQuickChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.). Primers are shown in Table 1. Each SULT1E1 cDNA was amplifiedby PCR and subcloned into the EcoRI restriction site of the eukaryoticexpression vector pCR3.1. After subcloning, all inserts were sequencedto assure that no spurious nucleotide point mutations had beenintroduced during the PCR amplifications.

COS-1 cells were transfected with 7 μg of each expression constructusing the TransFast™ reagent (Promega, Madison, Wis.) as suggested bythe manufacturer (i.e., using a 1:1 charge ratio). As a control, atransfection also was performed with 7 μg “empty” pCR3.1, i.e., vectorlacking an insert, to make it possible to correct for endogenous COS-1cell SULT activity. Seven μg of the control plasmid pSV-β-galactosidase(Promega, Madison, Wis.) was co-transfected with each SULT1E1 constructto make it possible to correct for transfection efficiency. Twoindependent transfections, each consisting of three separate plates,were performed with each expression construct.

After 48 hours in culture, the transfected cells were harvested and highspeed supernatant (HSS) cytosol preparations were prepared as describedby Wood et al., (1994) Biochem. Biophys. Res. Commun. 198:1119-1127.Aliquots of these cytosol preparations were stored at −80° C. prior tothe assay.

Example 5 Enzyme Assays

The HSS preparations of recombinant SULT1E1 variant proteins describedabove were used for the activity studies without any furtherpurification. The protein concentration of each recombinant proteinpreparation was determined by the dye-binding method of Bradford withbovine serum albumin (BSA) as a standard. β-galactosidase activity ineach of the COS-1 HSS preparations was measured with the β-galactosidaseEnzyme Assay System (Promega, Madison, Wis.). SULT1E1 enzyme activitywas measured with an assay that involves sulfate conjugation of asulfate acceptor substrate, 17-β estradiol (E2), in the presence of[³⁵S]-3′-phosphoadenosine-5′-phosphosulfate (PAPS), the sulfate donorfor the reaction. See Campbell et al. (1987) Biochem. Pharmacol,36:1435-1446; Foldes and Meek (Biochim. Biophys. Acta, 327:365-374,1973) or Hernandez et al. (Drug Metab. Disposit., 20:413-422, 1992).

Briefly, 0.4 μM ³⁵S-PAPS were used as the sulfate donor with 0.05 μM E2as the sulfate acceptor substrate in 8 mM dithiothreitol, 1.25 mM MgCl₂,and 10 mM potassium phosphate buffer at pH 6.5. Blanks were samples thatdid not contain E2. Cytosol from COS-1 cells that had been transfectedwith empty pCR3.1 was used to correct for endogenous SULT activity.Because SULTs display profound substrate inhibition, E2 concentrationsthat ranged from 10⁻³ M to 10⁻⁸ M were tested with each recombinantallozyme to ensure that the assays were performed at E2 concentrationsthat yielded maximal activity for that allozyme. Enzyme activity wasexpressed as nanomoles (nmole) of sulfate conjugated product formed perhour of incubation and adjusted to a percentage of the wild type SULTenzyme activity (FIG. 4).

Apparent K_(m) values for each allozyme were determined with bothcosubstrates. Initial experiments used 10-fold serial dilutions of E2that ranged from 10⁻³ to 10⁻⁹ M. Apparent K_(m) values were calculatedby using the method of Wilkinson with a computer program written byCleland (see, Wilkinson (1961) Biochem J 80:324-332; and Cleland (1963)Nature 198:463-365). Maximal activity for all allozymes was found at E2concentrations near 100 nM. A second set of experiments was performed inthe presence of 0.4 μM PAPS using eight concentrations of E2 that variedfrom 3.1 nM to 400 nM. For determination of apparent K_(m) values forPAPS, seven concentrations of PAPS that ranged from 2.7 nM to 150 nMwere assayed in the presence of 50 nM E2.

Two of the three allozymes (Asp22Tyr and Ala32Val) exhibitedsignificantly reduced levels of enzyme activity relative to the wildtype SULT1E1 enzyme (FIG. 4). Although Ala32Val exhibited reducedactivity (68.5% of wild-type), the apparent K_(m) values of Ala32Val forestradiol and PAPS were comparable to those of the wild type SULT1E1enzyme (Table 5). In contrast, although Asp22Tyr exhibited significantlyreduced activity compared to the wild type enzyme (27% of wild-type),the apparent K_(m) values of Asp22Tyr for estradiol and PAPS were 6 and3.3 fold higher, respectively, than the wild type enzyme. The thirdallozyme (Pro253His) exhibited a level of activity that was similar tothat of the wild type SULT1E1 enzyme (FIG. 4). The apparent K_(m) valuesof Pro253His for estradiol and PAPS were approximately 2.5 and 2.8 foldhigher, respectively, than the wild type SULT1E1 enzyme (Table 5).

Enzyme thermal stability was measured by diluting COS-1 cell supernatantpreparations for each of the recombinant SULT1E1 allozymes andincubating them in a water bath for 15 minutes at temperatures rangingfrom 28° C. to 46° C. The samples were placed on ice immediately afterincubation. Aliquots of the same supernatant preparations also were kepton ice as controls. Enzyme activity was measured in both heated andunheated samples, and blank values were determined for each temperaturestudied. T₅₀ values (i.e., values for temperatures resulting in 50%thermal inactivation, were calculated using the GraphPad Prism computerprogram (GraphPad Software, Inc., San Diego, Calif.). These studiesrevealed that the Asp22Tyr and Ala32Val allozymes had reduced T₅₀ valuesas compared to the wild-type enzyme, although the decrease for theAla32Val allozyme was not statistically significant.

TABLE 5 Human SULT1E1 allozyme substrate kinetics and thermal stabilityE2 as Varied Substrate PAPS as Varied Substrate Thermal SULT1E1 ApparentK_(m) V_(max)/ Apparent V_(max)/ Stability Allozyme (nM) V_(max) K_(m) ×(100) K_(m)(nM) Vmax K_(m) × (100) T₅₀ (° C.) Wild type 30.0 ± 5.0^(a)26.8 ± 4.4 89.3 56.0 ± 2.9^(a) 27.2 ± 0.7^(a) 48.5 38.5 ± 0.92 Asp22Tyr 220 ± 30.0^(a)   4.7 ± 0.3^(b) 2.2  240 ± 9.2^(a)  1.9 ± 0.1^(a) 0.8 35.0 ± 0.32^(c) Ala32Val 44.0 ± 5.0^(a) 22.8 ± 6.6 51.7 65.0 ± 4.2^(a) 5.7 ± 0.1^(a) 8.8 36.7 ± 1.2  Pro253His 97.0 ± 7.0^(a)  58.6 ± 2.5^(b)60.4  180 ± 7.5^(a) 34.8 ± 0.2^(a) 19.3 38.4 ± 0.46 Values are expressedas mean ± SEM (n = 3). V_(max) is expressed as nmol/hr/β-galactosidaseunits. ^(a)indicates that all values differed significantly from eachother (P ≦ 0.04). ^(b)indicates that values differed significantly fromeach other (P ≦ 0.01) except for the wild type and Ala32Val allozymes.^(c)indicates that the value differed significantly from the wild typeand Pro253His allozymes (P ≦ 0.003).

Example 6 Western Blot Analysis

Quantitative Western blot analysis was performed using COS-1 cytosolicextracts containing recombinant SULT1E1 allozymes. The quantity ofextracts loaded on 12.5% acrylamide gels was adjusted for each allozymeextract, so that each lane contained an equal quantity ofβ-galactosidase activity, i.e., gel loading was corrected for variationin transfection efficiency. The level of immunoreactive protein wasmeasured using a rabbit polyclonal antibody directed against amino acids1-13 of SULT1E1, with a cysteine residue added at the carboxy terminus.Properties of this antibody have been described elsewhere. See, Aksoy etal., (1994) Biochem. Biophys. Res. Commun. 200:1621-1629. Bound antibodywas detected using the ECL system (Amersham Pharmacia, Piscataway,N.J.). An Ambis Radioanalytic Imaging System, Quant Probe Version 4.31(Ambis, Inc., San Diego, Calif.) was used to quantitate immunoreactiveprotein in each lane, and the data were expressed as a percent of theintensity of the control wild type SULT1E1 protein band on that gel.

The average levels of immunoreactive SULT1E1 proteins, corrected fortransfection efficiency, were correlated with the relative levels ofenzyme activity for all three of the variant allozymes (FIGS. 4 and 5).Thus, the decreased activity of Asp22Tyr could be attributed both toalterations in the level of protein and to alterations in substratekinetics (see Table 5 and FIG. 5). However, substrate kinetics for theAla32Val allozyme were only slightly different from those of thewild-type enzyme. Thus, the decrease in enzyme activity for Ala32Valappeared to result primarily from a decreased level of immunoreactiveprotein. Although there were no differences in levels of either enzymeactivity or immunoreactive protein for Pro253His, this allozyme diddisplay significant increases in both K_(m) and V_(max) when comparedwith the wild-type enzyme—differences that may have “offset” each otherin their effects on enzyme activity.

Example 7 SULT1E1 Polymorphisms and Crystal Structures

The x-ray crystal structure of mouse SULT1E1 has been solved in thepresence of 3′-phosphoadenosine 5′-phosphate at a resolution of 2.5 Å(Kakuta et al. (1997) Nature Struct. Biol. 4:904-908). Recently,Pederson et al. (Pederson et al. (2002) J. Biol. Chem. 277:17928-17932)also solved the crystal structure of human SULT1E1 and reported that thesubstrate binding pocket of human SULT1E1 is very similar to that of themouse enzyme. The Asp22Tyr polymorphism disclosed herein was located 14residues downstream from the E2 substrate binding region, a conservedSULT “Region I” sequence motif (Weinshilboum and Otterness,“Sulfotransferase enzymes.” In: Kaufmann (ed.),Conjugation-Deconjugation Reactions in Drug Metabolism and Toxicity,chapter 2, “Handbook of Experimental Pharmacology” series, volume 112,pp. 45-78. Berlin Heidelberg: Springer-Verlag, 1994; Varin et al. (1995)Proc. Natl. Acad. Sci. USA 89:1286-1290; and Weinshilboum et al. (1997)FASEB J. 11:3-14). Since that polymorphism affected a residue located atthe entrance to the substrate binding pocket, the change in amino acidmight influence access of the substrate to the active site—a possibleexplanation for the observed increase in apparent K_(m) value (Table 5).In contrast, the Ala32Val polymorphism resulted in both decreased enzymeactivity and decreased immunoreactive protein (FIGS. 4 and 5), but itdid not affect substrate kinetics (Table 5). On the basis of the x-raycrystal structure, that amino acid was located on the surface of theprotein outside the putative substrate binding site. The third change inamino acid, Pro253His, was located 3 amino acids upstream of the “RegionIV” conserved SULT sequence motif, the PAPS binding site (Weinshilboumand Otterness, supra; Varin et al., supra; and Weinshilboum et al.,supra). This final polymorphism resulted in a 2 to 3 fold increase inK_(m) and V_(max) values for both E2 and PAPS (Table 5).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated human sulfotransferase 1E1 (SULT1E1) polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:7, wherein theamino acid sequence has a variation at residue 32 of SEQ ID NO:7.
 2. Theisolated SULT1E1 polypeptide of claim 1, wherein the amino acid sequencevariant at residue 32 of SEQ ID NO:7 is a valine substitution foralanine.
 3. The isolated polypeptide of claim 1, whereinsulfotransferase activity of the polypeptide is altered relative to awild type SULT1E1 polypeptide.